Polypeptide growth factors are hormone-like modulators of cell proliferation and differentiation. Growth factors are responsible for the regulation of a variety of physiological processes, including development, regeneration, and wound repair, and have been associated with normal as well as with pathophysiological processes. Numerous growth factors have been identified in various tissues and cells, and names that have been applied to these factors include epidermal growth factor, platelet-derived growth factor, nerve growth factor, hematopoietic growth factors, and fibroblast growth factor.
Fibroblast growth factor (FGF) was first described as an activity derived from bovine brain or pituitary tissue which was mitogenic for fibroblasts and endothelial cells. It was later noted that the primary mitogen from brain was different from that isolated from pituitary. These two factors were named acidic and basic FGF, respectively, because they had similar biological activities but differed in their isoelectric points. Acidic and basic FGF are proteins containing approximately 154 amino acids. Their amino acid sequences are related, with approximately 55% sequence identity between them.
Acidic and basic fibroblast growth factors are now known to be members of a larger family of heparin-binding growth factors that collectively trigger a variety of biological responses in many cell types, including those of mesoderm and neuroectoderm origin, such as endothelial cells, smooth muscle cells, adrenal cortex cells, prostatic and retina epithelial cells, oligodendrocytes, astrocytes, chrondocytes, myoblasts, and osteoblasts. As original family members, acidic and basic FGF are now denoted FGF-1 and FGF-2, respectively. Seven other members of the family have been identified on the basis of their modulation of cell proliferation and differentiation, and their sequence homology to other FGFs.
In addition to eliciting a mitogenic response that stimulates cell growth, fibroblast growth factors can stimulate a large number of cell types to respond in a non-mitogenic manner. These activities include promotion of cell migration into wound areas (chemotaxis), initiation of new blood vessel formulation (angiogenesis), modulation of nerve regeneration and survival (neurotrophism), modulation of endocrine functions, and stimulation or suppression of specific cellular protein expression, extracellular matrix production and cell survival (Baird, A., and Bohlen, P., Handbook of Exp. Pharmacol. 95(1): 369-418, Springer, 1990). These properties provide a basis for using fibroblast growth factors in therapeutic approaches to accelerate wound healing, nerve repair, collateral blood vessel formation, and the like. For example, fibroblast growth factors have been suggested to minimize myocardium damage in heart disease and surgery (U.S. Pat. No. 4,378,347 to Franco).
Current research regarding FGF-2 and other FGFs has centered on the molecular details of the receptor-mediated pathways by which their diverse physiological activities are expressed, as a way to gain information for the design of therapeutically useful agents that can either mimic or inhibit the action of these factors. Since the primary structure of FGF-2 isolated from a variety of sources is known, and bovine and human FGF-2 have been cloned and expressed in E. coli and S. cervisiae, recent attention has focused on secondary and tertiary structure.
The 3-dimensional structures of FGF-1 and FGF-2 have been determined (Eriksson, E. A., et al., Proc. Nat. Acad. Sci. U.S.A. 88: 3441-3445 (1991), Zhang, J., et al., Proc. Nat. Acad. Sci. U.S.A. 88: 3446-3450 (1991), and Zhu, H., et al., Science 251: 90-93 (1991)). In these studies, FGF-1 and FGF-2 were shown to exhibit a folding pattern strikingly similar to that observed for the cytokine interleukin-1.alpha., and interleukin-1.beta. (IL-1.alpha. and IL-1.beta.), protein factors produced by macrophages and T-cells in response to antigenic or mitogenic stimulation, though the primary structures of interleukin-1 polypeptides have only about a 10% amino acid sequence correspondence to FGFs.
The overall structure of FGF-2 can be described as a trigonal pyramid where each of the three sides are built of two .beta.-strands together forming a .beta.-sheet barrel of six antiparallel strands (Eriksson, E. A., et al., Proc. Nat. Acad. Sci. U.S.A. 88: 3441-3445 (1991)). The base of the pyramid is built of six additional .beta.-strands extending from the three sides of the pyramid to close one end of the barrel for a total of twelve .beta.-strands. Thus, a threefold repeat is observed in the folding of the polypeptide chain and a pseudo-three-fold axis passes through the center of the base of the molecule and extends through the apex of the pyramid (ibid.). Of the amino acids conserved within the FGF family of proteins, most are located within the core .beta.-strand regions of FGF-2, supporting the expectation that each of these proteins has an overall 3-dimensional structure similar to that of FGF-2.
The biological responses of FGF are mediated by the heparan sulfate-dependent binding of the growth factor to specific cell surface receptors (Givol, D., and Yayon, A., FASEB J. 6: 3362-3369 (1992) and Jaye, M., et al., Biochim. Biophys. Acta 1135: 185-199 (1992)), yet the molecular interactions of heparin and receptor with FGF and the exact nature of the events of the signal transduction pathway are unknown. Studies employing synthetic peptides related to the FGF sequence showed that FGF-2 (33-77) and (106-129) bind to heparin and act as weak partial agonists and antagonists in a mitogenic assay of FGF activity (Baird, A., et al. Proc. Nat. Acad. Sci. U.S.A. 85: 2324-2328 (1988)). The same study identified a sequence, FGF-2 (115-124), involved in receptor binding. The segment begins in the middle of the ninth .beta.-strand, makes a somewhat open loop on the surface of the folded molecule, and terminates at the beginning of the tenth .beta.-strand. This sequence (118-122) in the native protein forms a small surface loop that is close to a cluster of basic surface residues that may form a putative heparin binding site (Zhang, J., et al., Proc. Nat. Acad. Sci. U.S.A. 88: 3446-3450 (1991)).
This sequence also contains Thr-121, which can be phosphorylated by a cAMP-dependent protein kinase (Feige, J. J., and Baird, A., Proc. Nat. Acad. Sci. U.S.A. 86: 3174-3178 (1989)); Thr-121 is denoted in the paper as Thr-112 since the investigators employed the N-terminally truncated form of the polypeptide, which exhibits full biologic activity but has 146 rather than the usual 154 amino acids). Phosphorylation of Thr-121 results in the generation of a form of the protein that exhibits an increased capacity to compete with radiolabelled FGF-2 binding to its receptor, but no difference in the biological properties of the phosphorylated and non-phosphorylated forms of the protein were observed (ibid.). In contrast to FGF-2, FGF-1 is not a substrate for the kinase. The data are consistent with the hypothesis that the sequence 115-124 is involved in receptor binding, but they do not define the complete receptor binding domain of the molecule, nor do they demonstrate the physiological significance of phosphorylation of Thr-121.
In addition to heparin and receptor binding regions, there is evidence that specific sequences in FGF influence ligand-induced signal transduction. For example, deletion of residues 27-32 of FGF-2 (Lys-Asp-Pro-Arg-Leu) or mutation of the basic residues Arg-118, Lys-119, Lys-128, and Arg-129 did not appear to affect the mitogenic activity of the protein, but eliminated activation of plasminogen activator gene expression (Eur. Pat. Ap. Pub. No. 363,675 to Bergonzoni, L., et al., and Presta, M., et al., Biochem. Biophys. Res. Com. 185: 1098-1107 (1992)).
At least four different fibroblast growth factor receptors (FGFR) have been identified (Werner, S., et al., Mol. Cell. Bio. 12: 82-88 (1992)), and functional differences between different receptor forms have been observed. Different FGF receptor forms derived from the same gene via alternative splicing have different ligand binding properties, and analogous splice variants from different FGF receptor genes bind different members of the FGF family (Johnson, D. E., and Williams, L. T., Adv. Can. Res. 60: 1-41 (1993)). Thus, fibroblast growth factor receptors exhibit a multitude of structural variants, and considerable cross-reactivity between receptors and their various ligands (Yayon, A., et al., EMBO J. 11: 1885-1890 (1992)). Though the carboxy-terminal region of the third immunoglobulin-like domain appears to be a structural element that defines specificity of different FGF members (Werner, et al., and Yayon, et al., cited above), the precise nature of FGF-receptor-heparin interactions and the protein residues involved have yet to be elucidated.